6-Aminonicotinamide Induces $G_1$ Arrest by Elevating $p27^{kip1}$ as well as Inhibiting cdk2, Cyclin E and p-Rb in IMR32 Neuroblastoma Cell Line

  • Engliez Souad Ahmad (Department of Biology, College of Sciences, Dongguk University) ;
  • Park In-Kook (Department of Biology, College of Sciences, Dongguk University)
  • 발행 : 2005.12.01

초록

The effects of 6-aminonicotinamide (6-AN) on viability of IMR32 neuroblastoma cells in the presence of ATP or $NAD^+$ have been investigated. 6-AN caused marked reduction in cell viability and similar observations were also made with cells treated with 6-AN+ATP. However, cells treated with $6-AN+NAD^+$ showed cell viability similar to untreated cells. Morphologically, 6-AN and 6-AN+ATP treated cells showed loss of neurites, polyhedric shapes, shrinkage of cell bodies and formation of lysed cells, while $6-AN+NAD^+$ cells did not show any such changes. The flow cytometry analysis demonstrated that 6-AN increased cell population in $G_0/G_1$ phase and decreased cell population in Sand $G_2/M$ phase following a 72 h exposure. Western blot analysis showed that 6-AN stimulated a substantial increase in the level of the cdk inhibitor $p27^{kip1}$, but lowered the levels of cdk2, cyclin E and p-Rb. However, cdc25A and p53R2 were not significantly affected. Immunofluorscence staining of $p27^{kip1}$, cdk2, cyclin E and p-Rb revealed close correlation between the signal observed in the Western blot analysis. 6AN+ATP treated cells showed similar results obtained with 6-AN treated cells in expression of cdk2, cyclin E, p-Rb proteins and $p27^{kip1}$, $6-AN+NAD^+$ cells showed greater expression of cdk2, cyclin E and p-Rb than those in 6-AN and 6-AN+ATP treated cells. The results suggest that 6-AN induced the $G_0/G_1$ phase arrest in IMR32 neuroblastoma cell lines through the increase of $p27^{kip1}$ and the decrease of cdk2, cyclin E and p-Rb.

키워드

참고문헌

  1. Akiyama T, Yoshida T, Tsujita T, Shimizu M, Mizukami T, Okabe M, and Akinage S (1997) $G_1$ phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and cdk2 proteins as well as induction of cdk inhibitor p21 in p53-mutated human epidermoid carcinoma A431 cells. Cancer Res 57: 1495-1501
  2. Apasov S, Koshiba M, and Redegeld F (1995) Role of extracellular ATP and P1 and P2 classes of purinergic receptors in T-cell development and cytotoxic T lymphocyte effector functions. Immunol Rev 146: 5-19 https://doi.org/10.1111/j.1600-065X.1995.tb00680.x
  3. Belfi CA, Chattterjee DM, Gosky SJ, Berger SJ, and Berger NA (1990) Increased sensitivity of human colon cancer cells to DNA cross-linking agents after GRP78 up-regulation Biochem Biophys Res Commun 257: 361-368 https://doi.org/10.1006/bbrc.1999.0472
  4. Bielicki L and Krieglstein J (1976) Decreased GABA and glutamate concentration in rat brain after treatment with 6-aminonicotinamide. Naunyn-Schmiedeberg's Arch Pharmacol 294: 157-160 https://doi.org/10.1007/BF00507848
  5. Chow SC, Kass GE and Orrenius S (1997) Purines and their roles in apoptosis. Neuropharmacology 36: 1149-1156 https://doi.org/10.1016/S0028-3908(97)00123-8
  6. Estrela JM, Obrador E and Navarro J (1995) Elimination of Ehrlich tumours by ATP-induced growth inhibition, glutathione depletion and X-rays. Nature Med 1: 84-88 https://doi.org/10.1038/nm0195-84
  7. Fero ML, Rivkin M, Tasch M, Porter P, Carow CE, Firpo E, Polyak K, Tasai LH, Broudy V, Perlmutter RM, Kaushansky K, and Robert JM (1996) A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis and female sterility in $p27^{kip1}$ deficient mice. Cell 85: 733-744 https://doi.org/10.1016/S0092-8674(00)81239-8
  8. Ferrari D, Chiozzi P, and Falzoni s (1997) ATP-mediated cytotoxicity in microglial cells. Neuropharmacology 36: 1295-1301 https://doi.org/10.1016/S0028-3908(97)00137-8
  9. Genevieve R, Alessia M, Lucia DI, Pholippe C, Giulio F, Michele P, and Sylvain M. (2001) p27 cytoplasmic localization is regulated by phosphorylation on Ser10 and is not a prerequisite for its proteolysis. EMBO J 20: 6672-6682 https://doi.org/10.1093/emboj/20.23.6672
  10. Griffiths IR, Kelly PA, and Grome JJ (1981) Glucose utilization in the central nervous system in the acute gliopathy due to 6-aminonicotinamide. Lab Invest 44: 47-552
  11. Herken H, Lange K, and Kolbe H (1969) Brain disorders induced by pharmacological blockade of the pentose phosphate pathway. Biochem Biophys Res Commun 36: 93-100 https://doi.org/10.1016/0006-291X(69)90654-8
  12. Hunting D, Gowans B, and Henderson JF (1985) Effects of 6-Aminonicotinamide on cell growth, poly(ADP-ribose) synthesis and nucleotide metabolism. Biochem Pharmacol 34: 3999-4003 https://doi.org/10.1016/0006-2952(85)90379-X
  13. Iglesias JR and Iglesias RE (1974) Histochemical changes in the rat spinal cord after exposure to 6-aminonicotinamide. Acta Neuropathol 28: 223-232 https://doi.org/10.1007/BF00719027
  14. Jian-Ying LI, Xiao-Zhong W, Feng-Lin C, Jie-Ping YU, and He-Sheng L (2003) Nimesulide inhibits proliferation via induction of apoptosis and cell cycle arrest in human gastric adenocarcinoma cell line. World J Gastroenterol 9: 915-920 https://doi.org/10.3748/wjg.v9.i5.915
  15. Johnson WJ and McColl JD (1955) 6-Aminonicotinamide-a potent nicotinamide antagonist. Science 8: 122: 834 https://doi.org/10.1126/science.122.3174.834
  16. Kevin GM, Masatoshi T, and Michael B (2004) $NAD^+$ modulates p53 DNA binding specificity and function. Mol Cell Biol 24: 9958-9967 https://doi.org/10.1128/MCB.24.22.9958-9967.2004
  17. Kim JY and Park IK (1998) Effects of 6-aminonicotinamide on levels of soluble proteins and enzyme activities in various tissues of Japanese quail. Int J Biochem Cell Biol 30: 1337-1344 https://doi.org/10.1016/S1357-2725(98)00102-2
  18. Knoll-Kohler E, Wonjnorowicz F, and Sarkander HJ (1980) Correlated changes in neuronal cerebral rat brain RNA synthesis by 6-aminonicotinamide. Exp Brain Res 38: 173-179 https://doi.org/10.1007/BF00236738
  19. Lee YB and Park IK (2001) Effects of neurotoxin 6-aminonicotinamide on levels of enzyme activities and metabolites on quail plasma. Int J Biochem Cell Biol 33: 613-620 https://doi.org/10.1016/S1357-2725(01)00036-X
  20. Lukas J, Herzinger T, Hansen K, Moroni MC, Resnitzky D, Helin K, Reed SI, and Bartek J (1997) Cyclin E-induced S phase without activation of the p-Rb / E2F pathway. Genes Devel 1: 1479-1492 https://doi.org/10.1101/gad.11.11.1479
  21. Martin DS, Stolfi RL, Colofiore JR, and Nord LD (1996) Marked enhancement vivo of paclitaxel's tumor-regressing activity by ATP depleting modulation. Anticancer Res 7: 655-659 https://doi.org/10.1097/00001813-199608000-00006
  22. Mina R, Philip M, Joseph TN, and Geoffrey B (2002) ATP regulates the differentiation of mammalian skeletal muscle by activation of a P2X5 receptor on satellite cells. J Cell Biol 158: 345-355 https://doi.org/10.1083/jcb.200202025
  23. Ogata S, Takeuchi, M, Fujita H, Shibata K, Okumura K, and Taguchi H (2000) Apoptosis induced by nicotinamide-related compounds and quinolinic acid in Hl-60 cells. Biosci Biotechnol Biochem 64: 327-332 https://doi.org/10.1271/bbb.64.327
  24. Ohtsubo M, Theodoras A, Schumacher J, Roberts M, and Pagano M (1995) Human cyclin E, a nuclear protein essential for the $G_1$ to S phase transition. Mol Cell Biol 15: 2612-2624
  25. Ouwehand K, De Ruijter AJ, Bree CV, Caron HN, and Vankuilenburg AB (2005) Histone deacetylase inhibitor BL1521 induces a $G_1$-phase arrest in neuroblastoma cells through altered expression of cell cycle proteins. FEBS Letters 579: 1523-1528 https://doi.org/10.1016/j.febslet.2005.01.058
  26. Pan MH, Chen WJ, Shiau SY, Ho CT, and Lin JK (2002) Tangeretin induces cell-cycle $G_1$ arrest through inhibiting cyclin-dependent kinases 2 and 4 activities as well as elevating cdk inhibitors p21 and p27 in human colorectal carcinoma cells. Carcinogenesis 23: 1677-1684 https://doi.org/10.1093/carcin/23.10.1677
  27. Pardee AB (1989) $G_1$ events and regulation of cell proliferation. Science 246: 603-608 https://doi.org/10.1126/science.2683075
  28. Park WH, Kim ES, Jung CW, Kim BK, and Lee YY (2003) Monensin-mediated growth inhibition of SNU-C1 colon cancer cells via cell cycle arrest and apoptosis. Int J Oncol 22: 377-382 https://doi.org/10.1200/JCO.2004.99.219
  29. Shapiro DM, Dietrich LS, and Shils ME (1956) Quantitative biochemical differences between tumor and host as a basis for cancer chemotherapy V. Niacin and 6-aminonicotinamide. Cancer Res 17: 600-604
  30. Sherr CJ (1996) Cancer cell cycles. Science 274: 1672-1677 https://doi.org/10.1126/science.274.5293.1672
  31. Sherr CJ (1993) Mammalian $G_1$ cyclins. Cell 73: 1059-1065 https://doi.org/10.1016/0092-8674(93)90636-5
  32. Singh SP, Lipman J, Goldman H, Ellis FH, Aizenman L, Cangi MG, Signoretti S, Chiaur DS, Pagano M, and Loda M (1998) Loss or altered subcellular localization of p27 in Barrett's associated adenocarcinoma. Cancer Res 58: 1730-1735
  33. Street JC, Mahmood U, Ballon D, Alfieri AA, and Koutcher JA (1996) $^{13}C\;and\;^{31}P$ NMR investigation of effect of 6-aminonicotinamide on metabolism of RIF-1 tumor cells in vitro. J Biol Chem 271: 4113-4119 https://doi.org/10.1074/jbc.271.8.4113
  34. Tadakazu A, Kazuyo S, Makiko S, Tatsuya T, and Shiro A (1999) $G_1$-checkpoint function including a cyclin-dependent kinase 2 regulatory pathway as potential determinant of 7-hydroxystaurosporine(UCN-01)- induced apoptosis and $G_1$-phas accumulation. J Cancer Res 90: 1364-1372
  35. Yang CY and Park IK (2000) Neurotoxin 6-aminonicotinamide affects levels of soluble proteins and enzyme activities in various tissues of golden hamsters. Int J Biochem Cell Biol 32: 549-556 https://doi.org/10.1016/S1357-2725(99)00150-8
  36. Yang ES and Kerry LB (2003) Vitamine D inhibits $G_1$ to S progression in LN-Cap prostate cancer cells through p27 stabilization and cdk2 mislocalization to the cytoplasm. J Biol Chem 278: 46862-46868 https://doi.org/10.1074/jbc.M306340200
  37. Zeitz M, Lange K, Keller K, and Herken H (1978) Effect of 6-Aminonicotinamide on growth and acetylcholinesterase activity during differentiation of neuroblastoma cells in vitro Naunyn Schmiedebergs Arch Pharmacol 305: 117-21 https://doi.org/10.1007/BF00508280